Fig 1.
TCEA3 is regulated by myogenin.
A Tcea3 mRNA expression in E14.5 tongue tissue was quantified by qRT-PCR. B. Tcea3 mRNA expression was quantified in 6W heart and hindlimb tissues by qRT-PCR. Data were normalized to the expression of Hprt1. C. TCEA3 expression increases upon differentiation. Time course analysis of Tcea3 mRNA expression in C2C12 cells subjected to differentiation for the specified number of days (D) by qRT PCR. UD represents undifferentiated. D. Cells as in C. were assayed by western blot with indicated antibodies. E. C2C12 cell lines were transfected with shRNA constructs against Myog (shMyog) or the scrambled control (scr) and assayed by western blot analysis with indicated antibodies. F. Schematic of the Tcea3 promoter. Transcription start site is labeled as +1. G—H. MYOG binds to the Tcea3 promoter. ChIP assays were performed on C2C12 cells differentiated for 2 days with antibodies against myogenin and primers against E boxes present in the Tcea3 promoter (-131 to -126) (G) and the first intron (+816 to +822) (H). I-J. Loss of MYOG inhibits RNAPII recruitment to the Tcea3 promoter. ChIP assays were performed on cells as in E. with antibodies against RPB1, the largest subunit of RNAPII and primers against E boxes present in the Tcea3 promoter (I) and first intron (J). All graphs represent three independent experiments. S.D. represents the error bars, **p<0.01, ***p<0.001.
Fig 2.
Depletion of TCEA3 impairs both proliferation and differentiation in C2C12 cells.
A. Independent C2C12 cell lines expressing shRNA against Tcea3 (shTcea3) and scrambled control (scr) were selected and analyzed by qRT-PCR. Data were normalized to the expression of Hprt1. B. Cells in A. were analyzed by western blot with the indicated antibodies. C. Tcea3 depleted cells were assayed for proliferation. D-E. Tcea3 depleted cells were differentiated for the indicated number of days and analyzed by qRT-PCR (D) and western blot assays (E).F. Tcea3 depleted cells were differentiated for six days (D6). Images are bright field at 100X. G. Immunohistochemistry with antibodies against myosin heavy chain (MHC) was performed on cells as in F. DAPI was used to stain the nuclei. H. Gene expression of Mylpf mRNA in a time course of differentiation for Tcea3 depleted cells and scr control. I. Restoration of TCEA3 restores differentiation in Tcea3 depleted cells. Tcea3 depleted cells were transfected with an expression construct for TCEA3 (pTCEA3), differentiated for two days and used for western blot analysis with the indicated antibodies. J. TCEA1 is not downregulated with the depletion of TCEA3. Cells in A. were analyzed by qRT-PCR for Tcea1 mRNA at the indicated timepoints. S.D. represents the error bars, ***p<0.001.
Fig 3.
A. Tcea3 depleted cells (shTcea3) and scr control were differentiated for four days and analyzed for MRF dependent gene expression by qRT-PCR. B. TCEA3 is not highly expressed in 10T1/2 cells. mRNA expression of TCEA1 and TCEA3 were assayed by qRT-PCR in 10T1/12 cells and C2C12 cells (UD). C. TCEA3 induces the activity of MYOG and MYOD1 on muscle-specific luciferase reporter constructs. 10T1/2 cells were transiently transfected with the indicated constructs. Values are represented with respect to a luciferase vector with no promoter (pGL3 basic). pGL3(+) represents a luciferase vector with an SV40 promoter. Lmod2-luc is a MRF dependent luciferase vector. D. TCEA3 enhances endogenous MRF target gene expression. 10T1/2 cells were transfected with expression constructs for MYOG, MYOD and TCEA3 as indicated and gene expression was determined by qRT-PCR. Data were normalized to the expression of Hprt1. E. TCEA3 modestly activates MYOD1. Cells in D. and undifferentiated C2C12 cells (UD) were assayed for MYOD1 mRNA by qRT-PCR. S.D. represents the error bars, **p<0.01 ***p<0.001.
Fig 4.
TCEA3 interacts with MYOG and MYOD1.
A-B. Expression constructs for TCEA3 and MYOG or MYOD1 were co-transfected into HEK293 cells and immunoprecipitated with antibodies against MYOG (A) or MYOD1 (B). Immunoprecipitation was detected with antibodies against TCEA3. Cell extract is labeled Ext. C. Differentiated C2C12 cell extract (D4) was immunoprecipitated with antibodies against MYOG and detected with antibodies against TCEA3 and MYOG. D. TCEA3 binds to MRF dependent genes. ChIP assays were performed with antibodies against TCEA3 and primers to MRF dependent promoters as indicated. E. Myogenin recruits TCEA3 to promoters. ChIP assays were performed on C2C12 cells depleted for Myog (shMyog) and scr control with antibodies against TCEA3, MYOG and MYOD1. S.D. represents error bars and #p <0.05 (vs IgG) **p<0.01, ***p<0.001 as indicated. F. TCEA3 depletion decreases the binding of myogenin on a muscle-specific gene promoter. ChIP assays were performed in C2C12 cells depleted for Tcea3 (shTCEA3) and scr control with antibodies against TCEA3, MYOG and MYOD1. S.D. represents error bars and #p <0.05 (vs IgG) **p<0.01, ***p<0.001 as indicated.
Fig 5.
TCEA3 binds to RNAPII, promotes the recruitment of RNAPII and travels with elongating RNAPII.
A. TCEA3 binds to RNAPII. Cell extracts from C2C12 cells differentiated for 2 days (D2) were immunoprecipitated with antibodies against RPB1, the largest subunit of RNAPII. The western blot was probed with antibodies against TCEA3 and RPB1. Cell extract is labelled Ext. B. ChIP assays were performed on proliferating C2C12 cells (UD) with antibodies against TCEA3 and RNAPII and detected with primers corresponding to the promoter, intron-1 and intron-5 of Tnni2. S.D. represents error bars and #p <0.05 (vs IgG). C. ChIP assays were performed as in B. on C2C12 cells following differentiation for 6 days (D6). D. TCEA3 promotes the recruitment of RNAPII. ChIP assays were on C2C12 cells depleted for Tcea3 or scr control following 2 days of differentiation (D2) with antibodies against TCEA3 and RNAPII and primers against the Tnni2 promoter. S.D. represents error bars #p<0.05(vs IgG) **p<0.01, ***p<0.001 as indicated.
Fig 6.
TCEA3 translocates to the nucleus upon differentiation.
A. TCEA3 localization in C2C12 cells during proliferation (UD) and after 4 days of differentiation (D4). Immunofluorescence was performed with antibodies against TCEA3. Nuclei were stained with DAPI. Images were taken at 400x magnification. Scale bars represent 25 μm. B. Both nuclear (NE) and cytoplasmic (CE) fractions of proteins were extracted from proliferating (UD) and differentiated (D6) C2C12 cells and probed for TCEA3, LAMIN A/C (loading control for nucleus), TUBULIN (loading control for cytoplasm) and ACTIN. C. Model for the function of TCEA3 in myogenesis. During early differentiation, TCEA3 levels are low and TCEA3 is present in the nucleus and cytoplasm. Upon differentiation, MYOG levels increase and induce Tcea3, which translocates to the nucleus and enhances MRF driven gene expression by promoting RNAPII recruitment and elongation.